Quantitative detection method for cadmium sulfide in tetrahymena culture system

Abstract

The invention belongs to the technical field of cadmium sulfide detection, and particularly relates to a quantitative detection method for cadmium sulfide in a tetrahymena culture system. The method comprises the following steps: adding cadmium ions into tetrahymena to form a tetrahymena culture system sample, crushing, carrying out ultrafiltration, separating cadmium in the sample into free cadmium ions (Cd < 2 + >) and cadmium sulfide (CdS), and determining the content of cadmium in the separated liquid sample by using a high-sensitivity detection technology (such as an inductively coupled plasma mass spectrometer). Therefore, the contents of Cd < 2 + > and cadmium converted into CdS are respectively obtained. The method not only can provide composition information of cadmium in tetrahymena, but also can dynamically monitor the conversion efficiency of cadmium ions to cadmium sulfide, and provides reliable data support for evaluating the remediation effect of tetrahymena on cadmium pollution. In addition, the method has the advantages of simplicity and convenience in operation, high sensitivity, good repeatability and the like.

Description

Technical Field

[0001] The present invention belongs to the technical field of cadmium sulfide detection, and particularly relates to a method for quantitatively detecting cadmium sulfide in a Tetrahymena culture system. Background Art

[0002] Cadmium is a heavy metal pollutant with extremely strong toxicity and a relatively long biological half-life. Cadmium in water is easily absorbed by crops such as rice and accumulates step by step through the food chain, ultimately flowing to animals and humans. Since cadmium has a slow metabolism in organisms, even a small amount of continuous intake will gradually accumulate in the human body, leading to a series of serious diseases such as kidney damage. Even at low concentrations, cadmium pollution in water may cause long-term and irreversible harm to human health and the environment. Therefore, effective remediation measures must be taken, such as phytoremediation, chemical passivation, biosorption and other technologies, to reduce cadmium pollution in the water environment and protect human health and ecological safety.

[0003] At present, microbial remediation technology has attracted much attention from researchers in the field of heavy metal pollution control due to its significant advantages such as economic efficiency, strong specificity, and environmental friendliness. This technology can not only reduce the toxicity of cadmium through the metabolic activities of microorganisms, but also improve the remediation efficiency by genetically engineering organisms. As a model protozoan in toxicological research, Tetrahymena has shown some potential in the response and treatment of heavy metal environments. Research has shown that Tetrahymena can convert highly toxic cadmium ions (Cd 2 +) in the environment into low-toxic cadmium sulfide (CdS) through related metabolic pathways (including the cysteine synthesis pathway, glutathione metabolism pathway, etc.), thereby significantly reducing the toxicity of cadmium (Hongrui Lv. Research on the Cysteine Synthesis Pathway of Tetrahymena thermophila and Its Molecular Mechanism of Alleviating Heavy Metal Stress [D]. Shanxi University, 2021; Jiawei Tu, Tian Li, Zihan Gao, Jie Xiong, Wei Miao. Construction of CdS-Tetrahymena thermophila hybrid system by efficient cadmium adsorption for dye removal under light irradiation [J]. Journal of Hazardous Materials, 2022, 439, 129683). In addition, Tetrahymena can also adsorb heavy metals through various mechanisms such as endocytosis and metal-binding proteins, further reducing the bioavailability of cadmium in the environment. Therefore, Tetrahymena not only has high efficiency and environmental friendliness in cadmium pollution remediation, but also provides an important basis for the development of bioremediation technology.

[0004] Therefore, it is necessary to use the content of cadmium sulfide (CdS) generated by cadmium enrichment in Tetrahymena as a key indicator to evaluate the conversion rate of cadmium ions in Tetrahymena. However, there is no dedicated instrument or detection method on the market that can directly detect the CdS content in Tetrahymena, which to a certain extent limits the in-depth study of the evaluation of the cadmium conversion rate in Tetrahymena. To solve this problem, it is urgent to develop and establish an analytical method for quantitatively detecting the CdS content in the Tetrahymena culture system, which can provide reliable data support for the evaluation of the cadmium conversion rate in Tetrahymena. Summary of the Invention

[0005] To solve the above technical problems existing in the prior art, the present invention proposes a method for quantitatively detecting cadmium sulfide in a Tetrahymena culture system. This method forms a sample of the Tetrahymena culture system by adding cadmium ions to Tetrahymena. After fragmentation, ultrafiltration is used to separate the cadmium in the sample into two parts: free cadmium ions (Cd 2 +) and cadmium sulfide (CdS). A high-sensitivity detection technique (such as inductively coupled plasma mass spectrometry) is used to measure the cadmium content in the separated liquid sample, so as to obtain the contents of Cd 2 + and CdS respectively. This method can not only provide the compositional information of cadmium in Tetrahymena, but also dynamically monitor the efficiency of the conversion of cadmium ions to cadmium sulfide, providing reliable data support for evaluating the remediation effect of Tetrahymena on cadmium pollution. In addition, this method has the advantages of simple operation, high sensitivity, and good repeatability.

[0006] A method for quantitatively detecting the content of cadmium sulfide in a Tetrahymena culture system includes the following steps:

[0007] (1) Cultivation and treatment of Tetrahymena

[0008] Inoculate Tetrahymena into SPP medium containing 1% penicillin-streptomycin (preferably with an initial density of 3125 cells / mL), and culture it in a constant temperature shaking incubator at 20 - 35°C until a Tetrahymena culture system in the late stable stage is obtained (preferably culture at 135 r / min for 2 d, and the density is about 1500000 cells / mL at this time); wash the Tetrahymena culture system with Tris buffer (preferably Tris buffer with a concentration of 10 mM and pH 7.4), and resuspend it in SPP medium containing 1% penicillin-streptomycin to obtain a Tetrahymena culture system;

[0009] (2) Preparation and treatment of the sample:

[0010] (2-1) Add a CdCl2 solution with a certain concentration to the Tetrahymena culture system obtained in (1), and place it in a constant temperature shaking incubator at 20 - 35°C for slow and uniform shaking; collect Tetrahymena from the culture system as a sample as needed;

[0011] (2-2) Use an ultrasonic crusher to ultrasonically crush the sample. Add an equal volume of EDTA-2Na solution to the crushed sample and mix well. Use EDTA-2Na to fully chelate the cadmium ions adsorbed in the sample;

[0012] (3) Ultrafiltration of the sample: After adding ultrapure water to the sample treated with EDTA-2Na, add it to an ultrafiltration tube (15 ml, 50 kDa MWCO, product number UFC905096) for ultrafiltration. After ultrafiltration, collect the ultrafiltrate in the centrifuge tube;

[0013] (4) Sample digestion: Respectively mix the sample treated with EDTA-2Na in (2-2) and the ultrafiltrate collected in (3) with electronic grade nitric acid, add them to the digestion tube, tighten the lid, and place them in a microwave digester for digestion. Then place them in an acid evaporation instrument to evaporate the acid. After rinsing with nitric acid, collect the resulting solution;

[0014] (5) Determination of sample content: Filter the digested sample through a 0.22 μm aqueous filter membrane. Measure the cadmium content in the sample with an inductively coupled plasma mass spectrometer. The measurement results of the ultrafiltrate sample and the sample treated with EDTA-2Na in (2-2) correspond to the cadmium ion content and total cadmium content in the system respectively. Calculate the cadmium sulfide content and cadmium conversion rate through formulas (1-1) and (1-2). Among them, the cadmium sulfide content is the amount of cadmium converted into cadmium sulfide in the total cadmium: Cadmium sulfide content (mg / L) = Total cadmium content (mg / L) - Cadmium ion content (mg / L) (1-1)

[0015]

[0016] Further, the concentration to ensure full chelation of EDTA-2Na solution in (2-2) is determined by the following method:

[0017] Operate according to the above quantitative detection method, except that: Replace (2) with the following steps:

[0018] (2a) Preparation and treatment of the sample:

[0019] Collect the Tetrahymena in (1) as the sample, ultrasonically crush it with an ultrasonic crusher, add a certain concentration of CdCl2 solution to the crushed sample, mix well, and then add an equal volume of a certain concentration of EDTA-2Na solution and mix well;

[0020] According to the concentration of the CdCl2 solution, set different concentration ranges of EDTA-2Na solution to perform the above operations, calculate the percentage of cadmium ion content to total cadmium content at different concentrations, that is, the cadmium recovery rate. The concentration when the cadmium recovery rate reaches 95% is used as the lowest concentration for full chelation;

[0021] Further, the Cd after adding the CdCl2 solution in step (2) 2+ has a concentration of 5 mg / L - 10 mg / L.

[0022] Furthermore, when the Cd 2+ concentration is 5 mg / L in (2a), 60 - 80 μM of EDTA-2Na solution (preferably 60 μM) is added, and when the Cd 2+ concentration is 10 mg / L, 120 - 180 μM of EDTA-2Na solution (preferably 120 μM) is added.

[0023] Further, the preparation of the SPP medium: The solvent is ultrapure water, and the solutes and their concentrations are as follows: glucose 2 g / L, peptone 20 g / L, yeast extract 1 g / L, ferric citrate 0.03 g / L; sterilize at 121 °C for 15 min and then set aside.

[0024] Further, the ciliate is Tetrahymena thermophila SB210.

[0025] Compared with the prior art, the present invention has the following advantages and beneficial effects:

[0026] The present invention has established a method for quantitatively detecting cadmium sulfide in a ciliate culture system. By adding cadmium ions to the ciliate to form a sample of the ciliate culture system, after breaking the system at different fixed time points as required, the cadmium in the sample is separated into Cd 2+ and CdS by ultrafiltration, and then the cadmium content of the separated sample is measured to calculate the dynamic Cd 2+ and CdS contents. This method can not only provide the compositional information of cadmium in the ciliate, but also dynamically monitor the conversion efficiency of cadmium ions to cadmium sulfide, providing reliable data support for evaluating the remediation effect of the ciliate on cadmium pollution. BRIEF DESCRIPTION OF THE DRAWINGS

[0027] Figure 1 is the main flow chart of the method for quantitatively detecting cadmium sulfide in the ciliate culture system provided by the present invention;

[0028] Figure 2 is the graph showing the change relationship between the EDTA-2Na concentration and the cadmium recovery rate under different cadmium ion concentrations in Example 1 (a graph with a cadmium ion concentration of 5 mg / L and b graph with a cadmium ion concentration of 10 mg / L);

[0029] Figure 3Graph showing the linear relationship between the added cadmium ion concentration and the actual cadmium ion concentration measured in the sample under different total cadmium concentrations in Example 2 (total cadmium concentration is 5 mg / L in Figure a and 10 mg / L in Figure b);

[0030] Figure 4 Trend graph of cadmium conversion rate of Tetrahymena thermophila at different time points and different cadmium ion concentrations in Example 3. Detailed implementation methods

[0031] The following examples are only used to specifically illustrate the implementation methods of the present invention, but they cannot be construed as limiting the protection scope of the present invention.

[0032] The Tetrahymena used is Tetrahymena thermophila SB210, provided by Cornell University, USA (https: / / tetrahymena.vet.cornell.edu / ).

[0033] Preparation of SPP medium: The solvent is ultrapure water, and the solutes and their concentrations are as follows: glucose 2 g / L, peptone 20 g / L, yeast extract 1 g / L, ferric citrate 0.03 g / L; sterilize at 121 °C for 15 min and set aside.

[0034] Method for preparing cadmium chloride stock solution: Dissolve 0.10157 g of cadmium chloride hemipentahydrate powder in 50 mL of water (final cadmium concentration is 1000 mg / L), and filter with a 0.22 μm filter membrane (Millex-GP 0.22 μm) to remove bacteria and particles.

[0035] Method for preparing EDTA-2Na stock solution: Dissolve 3.7224 g of disodium ethylenediaminetetraacetate dihydrate powder in 500 mL of water (final concentration is 20 mM), and filter with a 0.22 μm filter membrane to remove bacteria and particles.

[0036] Method for preparing Tris buffer: Weigh 1.2114 g of tris(hydroxymethyl)aminomethane (Tris) powder and dissolve it in 1 L of water (final concentration is 10 mM), adjust the pH to 7.4 with hydrochloric acid, and sterilize at 120 °C for 15 min.

[0037] The basic design principle of the present invention is:

[0038] EDTA-2Na solution can desorb the cadmium adsorbed by Tetrahymena cell debris through chelation, and through ultrafiltration, the desorbed cadmium can be separated into two parts: free cadmium ions (Cd 2 +) and cadmium sulfide (CdS). Then, a highly sensitive detection technology is used to measure the cadmium content in the separated liquid sample, so as to calculate the cadmium content converted into CdS.

[0039] To ensure the reliability of the determination results of the samples in the Tetrahymena culture system, an important influencing factor is the concentration of the EDTA-2Na solution. Because if the amount of EDTA-2Na is too small, the adsorbed cadmium cannot be desorbed sufficiently; if the amount of EDTA-2Na is too large, in addition to desorbing cadmium ions, a small amount of cadmium in cadmium sulfide will also be chelated out, which will affect the determination of the cadmium composition in the experimental samples. Therefore, in Example 1, the optimal EDTA-2Na concentration was explored under different cadmium ion concentration treatments.

[0040] Example 1 Establishment of the Tetrahymena culture system and investigation of the EDTA-2Na concentration conditions

[0041] (1) Cultivation and treatment of Tetrahymena: Thermophilic Tetrahymena was inoculated into SPP medium containing 1% penicillin-streptomycin, with an initial density of 3125 cells / mL. After culturing in a constant temperature shaker incubator at 30 °C at 135 r / min for 2 days, a thermophilic Tetrahymena culture system in the late stationary phase was obtained (the density of Tetrahymena was approximately 1500000 cells / mL); the thermophilic Tetrahymena culture system was washed twice with Tris buffer (10 mM, pH 7.4) and resuspended in fresh SPP medium containing 1% penicillin-streptomycin.

[0042] (2) Preparation and treatment of samples: Tetrahymena was collected from the culture system as a sample, and the sample was ultrasonically disrupted (50% power, 10 min) using an ultrasonic disruptor (model Ningbo Xinzhi JY92-IIN, 650 W, horn 10 mm). CdCl2 stock solution (cadmium concentration 1000 mg / L) was added to the disrupted sample to a final cadmium concentration of 5 mg / L or 10 mg / L. After mixing, an equal volume of EDTA-2Na solution was added, and the mixture was shaken for 10 min to mix evenly;

[0043] (3) Ultrafiltration of samples: Take 500 μL of the sample treated with EDTA-2Na above, add 2500 μL of ultrapure water, mix, and then add the mixture to the upper filter of an ultrafiltration tube (15 ml, 50 kDa MWCO, product number UFC905096). Centrifuge at 3000×g for 10 min using a centrifuge with a swinging bucket rotor. Add 2000 μL of ultrapure water to the ultrafiltration tube again and centrifuge once more. After ultrafiltration, collect all the ultrafiltrate in the centrifuge tube.

[0044] (4) Sample digestion: 2 mL of the sample without ultrafiltration treatment and the ultrafiltrate collected after ultrafiltration were respectively mixed with 5 mL of electronic-grade 2% nitric acid, added to a digestion tube, capped and tightened, and then placed in a microwave digestion instrument. Digestion was carried out according to a certain temperature-rising program (temperature-rising time: 40 min; digestion temperature: 180 °C; holding time: 15 min). After digestion, the digestion tube was taken out, the lid was opened, and it was placed in an acid-evaporating instrument to evaporate to less than 0.2 mL at 160 °C. The digestion tube was taken out, rinsed with 2% nitric acid, all the solutions were collected, transferred to a 15 mL centrifuge tube, and fixed volume to 10 mL with 2% nitric acid.

[0045] (5) Determination of sample content: The digested sample was filtered with a 0.22 μm aqueous filter membrane (Millex-GP 0.22 μm), and the filtered sample was directly used to determine the cadmium content in the sample by inductively coupled plasma mass spectrometry. According to the determination results of the ultrafiltrate sample and the sample without ultrafiltration treatment, the cadmium ion content and the total cadmium content in the system can be obtained respectively.

[0046] In this experiment, a total of 2 cadmium ion concentration systems and 8 corresponding EDTA-2Na concentration systems were set up, with a total of 16 groups, and each group had 3 parallels. The specific concentration settings are shown in Table 1.

[0047] Table 1 Cadmium ion concentration system and EDTA-2Na concentration settings

[0048]

[0049] The experimental results are as Figure 2 shown in Figure a. When the cadmium ion concentration is 5 mg / L and there is no EDTA-2Na, the cadmium recovery rate (the percentage of cadmium ion content to total cadmium content) is only about 30%; when the EDTA-2Na concentration is set to 10 μM, 20 μM, 30 μM, 50 μM, the cadmium recovery rate does not reach 100%, while when the EDTA-2Na concentration is set to 60 μM, 70 μM, and 80 μM, the cadmium recovery rate is close to 100%. In the experimental setup of subsequent Example 2, in order to saturate the cadmium recovery rate, when the cadmium ion or total cadmium concentration is 5 mg / L, the EDTA-2Na concentration used is 60 μM.

[0050] The experimental results Figure 2 shown in Figure b. When the cadmium ion concentration is 10 mg / L and there is no EDTA-2Na, the cadmium recovery rate is only about 20%; when the EDTA-2Na concentration is set to 40 μM, 60 μM, 80 μM, 100 μM, the cadmium recovery rate does not reach 100%, while when the EDTA-2Na concentration is set to 120 μM, 160 μM, and 180 μM, the cadmium recovery rate is close to 100%. In the experimental setup of subsequent Example 2, in order to saturate the cadmium recovery rate, when the cadmium ion or total cadmium concentration is 10 mg / L, the EDTA-2Na concentration used is 120 μM.

[0051] Methodological Investigation of Example 2

[0052] 2.1 Investigation of Linear Relationship

[0053] To investigate the practicality of this method, the cultivation and treatment of Tetrahymena were carried out according to the method in Example 1. The difference is that during the preparation and treatment of the sample, adding the CdCl2 stock solution to the broken sample was replaced with a certain amount of CdCl2 and cadmium sulfide (CdS), where the total cadmium concentrations were 5 mg / L and 10 mg / L respectively. Subsequently, the sample ultrafiltration, sample digestion, and sample content determination were still carried out according to the method in Example 1. Finally, the linear relationship between the added cadmium ion concentration and the actually measured cadmium ion concentration was compared.

[0054] To obtain a certain amount of total cadmium concentration, first, prepare a cadmium sulfide CdS suspension: fully mix the cadmium chloride solution and sodium sulfide solution, wash the precipitate three times with ultrapure water, then resuspend it in ultrapure water, and use an ultrasonic crusher to break it to obtain a homogeneous cadmium sulfide suspension. Carry out digestion and content determination of its concentration according to the method in Example 1, and then dilute it to a CdS stock solution with a cadmium concentration of 1000 mg / L.

[0055] Secondly, prepare mixtures of CdCl2 (Cd 2+ ) and cadmium sulfide (CdS) with different cadmium ion concentrations, and the total cadmium concentrations are 5 mg / L and 10 mg / L respectively, as shown in Tables 2 and 3. When the total cadmium concentration is 5 mg / L, the Cd 2+ concentration of No. A1 is 0 mg / L Cd 2+ , 5 mg / L CdS (in the following examples, the expression of the concentration of cadmium sulfide (CdS) represents the concentration of cadmium in CdS in the mixture), that is, add 5 μL of CdS stock solution (1000 mg / L) and 0 μL of CdCl2 (Cd concentration 1000 mg / L) to the broken Tetrahymena thermophila culture system, and make up the volume to 10 mL. For No. B1 - H1, the Cd 2+ concentrations are 0.5, 1, 2, 3, 4, 4.5, 4.8, 5 mg / L respectively, and the CdS concentrations are 4.5, 4, 3, 2, 0.5, 0.2, 0 mg / L respectively. When the total cadmium concentration is 10 mg / L, the Cd

[0056] concentrations of No. B2 - H2 are 0, 1, 2, 4, 6, 8, 9, 10 mg / L respectively, and the CdS concentrations are 10, 9, 8, 6, 4, 2, 1, 0 mg / L respectively. There are 16 groups of samples in total, with 3 parallels in each group.

[0057]

[0058] Table 3 Different cadmium concentration settings in the system with a total cadmium concentration of 10 mg / L

[0059]

[0060]

[0061] Using the cadmium ion concentration added to the sample (preset cadmium ion concentration) as the abscissa and the actually measured cadmium ion concentration (measured cadmium ion concentration) as the ordinate, the linear relationship was investigated. Figure 3 a Total cadmium concentration is 5 mg / L, Figure 3 b Total cadmium concentration is 10 mg / L. The results show that within the corresponding linear range, the correlation coefficients (R 2 ) are 0.9957 and 0.9964 respectively, and the cadmium ion concentration has a good linear relationship in both systems. However, as Figure 2 shown in a, when the preset Cd 2+ concentration is less than 3 mg / L (i.e., the preset CdS concentration is greater than 2 mg / L), EDTA-2Na will chelate a small part of the cadmium in CdS, making the measured Cd 2+ concentration slightly higher than the preset concentration; when the preset Cd 2+ concentration is greater than or equal to 3 mg / L (i.e., the preset CdS concentration is less than or equal to 2 mg / L), the measured Cd 2+ concentration is not much different from the preset concentration.

[0062] 2.2 Investigation of repeatability

[0063] To investigate the repeatability of this method, the cultivation and treatment of Tetrahymena, the preparation and treatment of samples were carried out according to the method of Example 1. The samples in D1, E1, D2, E2, and F2 of this Example 2.1 were prepared, and three samples of each number were prepared. Equal volumes of EDTA-2Na solutions with corresponding concentrations were added, shaken and mixed evenly for 10 min, and then the samples were ultrafiltered, digested, and the sample content was measured to obtain the cadmium ion content in the filtrate. The repeatability of the method was investigated by calculating the relative standard deviation (Relative Standard Deviation, RSD) between the three groups of measurement results.

[0064] The repeatability results are shown in Table 4. The RSD range of the cadmium content in the filtrate of each sample is 1.18% - 4.13%, and the RSD values are all less than 5%, indicating that the repeatability of the method is good.

[0065] Table 4 Results of the repeatability experiment

[0066]

[0067] 2.3 Investigation of stability

[0068] In order to investigate the stability of this method, the culture and treatment of Tetrahymena, the preparation and treatment of samples were carried out according to the method of Example 1, and the samples numbered D1, E1, D2, E2, and F2 in Example 2.1 were prepared and placed in a 4°C environment. After 0h, 12h, and 24h, an equal volume of EDTA-2Na solution of corresponding concentration was added, and the mixture was shaken for 10min, followed by subsequent sample ultrafiltration, sample digestion, and sample content determination to obtain the cadmium ion content in the filtrate. The stability of the method was investigated by calculating the relative standard deviation (RSD) between the sample determination results at three time points.

[0069] The determination results and relative standard deviations are shown in Table 5. The stability results show that the RSD range of the cadmium content in the filtrate of each sample is 0.88% to 3.54%, and the RSD values ​​are all less than 5%, indicating that the samples have good stability within 24 hours at 4°C.

[0070] Table 5 Stability test results

[0071]

[0072] Example 3 Evaluation of cadmium conversion rate under different treatment time of Tetrahymena thermophila

[0073] The above method was applied to thermophilic Tetrahymena to detect its cadmium conversion rate at different times. Tetrahymena was cultured and treated according to the method of Example 1, except that: during the preparation and treatment of the sample, after adding CdCl2 mother solution (1000 mg / L) to the Tetrahymena to the desired final concentration (5 mg / L or 10 mg / L), it was placed in a constant temperature shaking incubator at 30-35°C, and slowly shaken at a constant speed of 50 r / min. The Tetrahymena culture system was collected as a sample at different time points (including 0h, 3h, 6h, 12h, 24h, and 48h) for ultrasonic disruption, and an equal volume of EDTA-2Na (5 mg / L Cd 2+ Add equal volumes of 60 μM EDTA-2Na, 10 mg / L Cd 2+ An equal volume of 120 μM EDTA-2Na was added at a concentration of 1.0 μM. Then, the sample was ultrafiltered, the sample was digested, and the sample content was determined according to the method of Example 1. Such sample processing can accurately measure the cadmium conversion rate of Tetrahymena thermophila at different time points.

[0074] The cadmium sulfide content is calculated according to formula 1-1 (the cadmium sulfide content is the amount of cadmium converted into cadmium sulfide in the total cadmium).

[0075] Cadmium sulfide content (mg / L) = total cadmium content (mg / L) - cadmium ion content (mg / L) (1-1)

[0076] The cadmium conversion rate was calculated according to formula 1-2.

[0077]

[0078] The experimental results are as Figure 4 shown. The conversion rate of cadmium sulfide by Tetrahymena thermophila shows an increasing trend at different time points until it reaches a steady state, showing time dependence. When the Cd 2+ concentration is 5 mg / L, the conversion rate of cadmium approaches saturation at 12 h, and the conversion rate is 91.5%; when the Cd 2+ concentration is 10 mg / L, the conversion rate of cadmium approaches saturation at 24 h, and the conversion rate is 78.3%.

[0079] In summary, the present invention provides a method for quantitatively detecting cadmium sulfide in a Tetrahymena culture system, which has a good linear relationship, good repeatability and stability. This method is applicable to determining the content of cadmium sulfide in a Tetrahymena culture system and detecting the conversion rate of cadmium in terms of quantification.

[0080] It should be noted that in this application, relational terms such as first and second are only used to distinguish one entity or operation from another entity or operation, and do not necessarily require or imply any such actual relationship or order between these entities or operations. Moreover, the term "comprising", "including" or any other variant thereof is intended to cover non-exclusive inclusion, so that a process, method, article or device comprising a series of elements not only includes those elements, but also includes other elements not expressly listed, or further includes elements inherent to such process, method, article or device.

Claims

1. A method for quantitatively detecting the cadmium sulfide content in a Tetrahymena culture system, characterized in that, It includes the following steps: (1) Cultivation and treatment of Tetrahymena Inoculate Tetrahymena into SPP medium containing 1% penicillin-streptomycin, and culture it in a constant temperature shaking incubator at 20-35°C to obtain a Tetrahymena culture system in the late stable stage. After washing, resuspend it in SPP medium containing 1% penicillin-streptomycin to obtain a Tetrahymena culture system; (2) Preparation and treatment of samples: (2-1) Add a certain concentration of CdCl2 solution to the Tetrahymena culture system obtained in (1), and place it in a constant temperature shaking incubator at 20-35°C; then collect Tetrahymena from the culture system as samples as needed; (2-2) Ultrasonically crush the samples with an ultrasonic crusher, add an equal volume of EDTA-2Na solution to the crushed samples and mix well, and use EDTA-2Na to fully chelate the cadmium ions adsorbed in the samples to obtain the samples treated with EDTA-2Na; (3) Ultrafiltration of samples: After adding ultrapure water to the samples treated with EDTA-2Na, ultrafilter them in a 50k ultrafiltration tube, and collect the ultrafiltrate after ultrafiltration; (4) Digestion of samples: Mix the samples treated with EDTA-2Na in (2-2) and the ultrafiltrate collected in (3) with electronic grade nitric acid respectively, put them into a microwave digester for digestion, then place them in an acid-removing instrument to remove acid, wash with nitric acid, and collect the obtained solution; (5) Determination of sample content: Filter the digested samples with a water phase filter membrane, and measure the cadmium content in the samples with an inductively coupled plasma mass spectrometer. The measurement results of the ultrafiltrate samples and the samples treated with EDTA-2Na in (2-2) correspond to the cadmium ion content and total cadmium content in the system respectively. The cadmium sulfide content and cadmium conversion rate are obtained through formulas (1-1) and (1-2), where the cadmium sulfide content is the amount of cadmium converted into cadmium sulfide in the total cadmium: Cadmium sulfide content (mg / L) = Total cadmium content (mg / L) - Cadmium ion content (mg / L) (1-1) 2. The quantitative detection method according to claim 1, characterized in that When ensuring that the EDTA-2Na solution fully chelates the cadmium ions adsorbed in the samples in (2-2), the concentration of the EDTA-2Na solution is determined by the following method: Operate according to the quantitative detection method described in Claim 1, except that: Replace (2) with the following steps: (2a) Preparation and treatment of samples: Collect the Tetrahymena in (1) as samples, ultrasonically crush them with an ultrasonic crusher, add a certain concentration of CdCl2 solution to the crushed samples, mix well and then add an equal volume of a certain concentration of EDTA-2Na solution and mix well; According to the concentration of the CdCl2 solution, set different concentration ranges of EDTA-2Na solution to carry out the above operations, calculate the percentage of cadmium ion content and total cadmium content at different concentrations, that is, the cadmium recovery rate. The concentration when the cadmium recovery rate is 95% is used as the lowest concentration for full chelation.

3. The quantitative detection method according to claim 1 or 2, characterized in that, The Cd after adding the CdCl2 solution in the step (2) 2+ has a concentration of 5 mg / L - 10 mg / L.

4. The quantitative detection method according to claim 3, characterized in that, Cd in the above (2a) 2+ When the concentration of Cd is 5 mg / L, add 60 μM - 80 μM of EDTA-2Na solution. Cd 2+ When the concentration of Cd is 10 mg / L, add 120 μM - 180 μM of EDTA-2Na solution.

5. The quantitative detection method according to claim 1 or 2 or 4, characterized in that, The preparation method of the SPP medium is as follows: the solvent is ultrapure water, and the solutes and their concentrations are as follows: glucose 2 g / L, peptone 20 g / L, yeast extract 1 g / L, ferric citrate 0.03 g / L; sterilize at 121 °C for 15 min and then set aside.

6. The quantitative detection method according to claim 1 or 2 or 4 or 5, characterized in that The Tetrahymena is Tetrahymena thermophila SB210.

7. The quantitative detection method for the cadmium sulfide content in a Tetrahymena culture system according to claim 1 or 2, characterized in that, The method in (2-1) further includes separately collecting Tetrahymena from the culture system at fixed time points as samples according to requirements, keeping other steps unchanged, measuring the cadmium conversion rate of the samples at different time points, and realizing dynamic quantitative measurement of the cadmium sulfide content.

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